Synergistic effects of combined cisplatin and Clinacanthus nutans extract on triple negative breast cancer cells

Objective Triple negative breast cancer (TNBC) is the most invasive breast cancer subtype enriched with cancer stem cells. TNBCs do not express estrogen, progesterone, or human epidermal growth factor receptor 2 (HER2) receptors, making them difficult to be targeted by existing chemotherapy treatments. In this study, we attempted to identify the effects of combined cisplatin and Clinacanthus nutans treatment on MDA-MD-231 and MDA-MB-468 breast cancer cells, which represent TNBC subtypes. Methods The phytochemical fingerprint of C. nutans ethanolic leaf extract was evaluated by LC–MS/MS analysis. We investigated the effects of cisplatin (0–15.23 μg/mL), C. nutans (0–50 μg/mL), and a combination of cisplatin (3.05 μg/mL) and C. nutans (0–50 μg/mL), on cell viability, proliferation, apoptosis, invasion, mRNA expression in cancer stem cells (CD49f, KLF4), and differentiation markers (TUBA1A, KRT18) in TNBC cells. In addition, we also studied the interaction between cisplatin and C. nutans. Results Derivatives of fatty acids, carboxylic acid ester, and glycosides, were identified as the major bioactive compounds with potential anticancer properties in C. nutans leaf extract. Reductions in cell viability (0–78%) and proliferation (2–77%), as well as a synergistic anticancer effect, were identified in TNBC cells when treated with a combination of cisplatin and C. nutans. Furthermore, apoptotic induction via increased caspase-3/7 activity (MDA-MB-231: 2.73-fold; MDA-MB-468: 3.53-fold), and a reduction in cell invasion capacity to 36%, were detected in TNBC cells when compared to single cisplatin and C. nutans treatments. At the mRNA level, cisplatin and C. nutans differentially regulated specific genes that are responsible for proliferation and differentiation. Conclusion Our findings demonstrate that the combination of cisplatin and C. nutans represents a potential treatment for TNBC.

Objective: Triple negative breast cancer (TNBC) is the most invasive breast cancer subtype enriched with cancer stem cells. TNBCs do not express estrogen, progesterone, or human epidermal growth factor receptor 2 (HER2) receptors, making them difficult to be targeted by existing chemotherapy treatments. In this study, we attempted to identify the effects of combined cisplatin and Clinacanthus nutans treatment on MDA-MD-231 and MDA-MB-468 breast cancer cells, which represent TNBC subtypes.
Results: Derivatives of fatty acids, carboxylic acid ester, and glycosides, were identified as the major bioactive compounds with potential anticancer properties in C. nutans leaf extract. Reductions in cell viability (0 e78%) and proliferation (2e77%), as well as a synergistic anticancer effect, were identified in TNBC cells when treated with a combination of cisplatin and C. nutans. Furthermore, apoptotic induction via increased caspase-3/7 activity (MDA-MB-231: 2.73-fold; MDA-MB-468: 3.53-fold), and a reduction in cell invasion capacity to 36%, were detected in TNBC cells when compared to single cisplatin and C. nutans treatments. At the mRNA level, cisplatin and C. nutans differentially regulated specific genes that are responsible for proliferation and differentiation.
Conclusion: Our findings demonstrate that the combination of cisplatin and C. nutans represents a potential treatment for TNBC.

Introduction
Breast cancer is known for its heterogeneity and consists of numerous tumor cells ranging from stem cell-like cells to more differentiated cells that determine the fate of the disease. 1 A recent study by Sung et al. reported that the GLOBOCAN 2020 ranked female breast cancer as the most significant diagnosed cancer with a total number of 2.3 million new cases (11.7%) and the fifth ranked form of cancer in terms of mortality. 2 Triple negative breast cancer (TNBC) lacks three important hormone receptors (ER, PR and HER2) and is responsible for 20% of all cases of breast cancer. 3e6 Of the intrinsic subtypes of breast cancer, TNBC is considered the most lethal subtype due to its clinically aggressive behavior and the absence of targeted therapies. 3,7 In addition, TNBCs are known to be enriched with functional cancer stem-like cells which exhibit a high migration pattern and express some specific breast cancer genes which make these cells the most invasive subtype of all breast cancers. 8,9 In general, CSCs are able to self-renew and differentiate into various cell types, thus resembling the role of healthy stem cells. 8,9 Thus, this CSC sub-population is associated with chronic effects in various cancers including TNBCs. At present, the chemotherapeutic drugs that are used for the treatment of TNBC are often associated with significant toxicity and severe side effects. 10,11 In addition, the presence of CSCs in TNBC tumors is often associated with the development of chemotherapy resistance following chemotherapy thus complicating TNBC treatment 12 and leading to a highly metastatic and recurrent condition. Therefore, patients with TNBC are often difficult to treat and experience poorer survival rates when compared to patients with other breast cancer types. In recent years, TNBCs have shown specific sensitivity towards cisplatin, the first metal based anti-cancer drug. 1,13,14 Cisplatin is a metal-based anti-tumor drug that has been found to be effective in the treatment of various cancers. 14 This is a well-known cytotoxic drug that is claimed to be capable of interfering with DNA activity upon entering the nucleus of cells, thus preventing the DNA repair process and ultimately leading to cell death. 15 Nevertheless, recent studies have shown that cisplatin may exhibit other mechanisms of action, apart from apoptosis, such as inducing the differentiation of cancer cells. 1 Although previous studies reported that cisplatin can give rise to certain side effects following initial treatment, 16,17 accumulating evidence shows that the combination of cisplatin with other potential anticancer drugs can induce either autophagy or apoptosis in various types of cancer cells. 14,17,18 Cisplatin is also highly toxic to cancer cells with metastatic characteristics. 15 The Clinacanthus nutans (C. nutans) plant has emerged as an important traditional herb that represents a potential chemoprevention alternative for cancer patients. 19 C. nutans extracts contain various phytochemical compounds, including fatty acids, phenolics, glycosides, glycoglycerolipids, cerebrosides, and monoacylmonogalactosylglycerol, with useful biological capabilities. 20e26 Naturally-derived phytochemical constituents in C. nutans extracts also exhibit cytotoxicity effects through the induction of apoptosis 23 and antioxidant activity and could reduce the risk of cancer development. 20,24,25 Furthermore, C. nutans is preferred by individuals because it is a natural herb that is relatively safe with fewer side effects than conventional drugs. 26,27 In this study, we demonstrate the potential anticancer effects of a combination of cisplatin and C. nutans on MDA-MB-231 and MDA-MB-468 cells at the cellular and molecular levels.
The cells were maintained in T25 flasks (SPL Lifesciences, Korea) at 37 C and 5% CO 2 . The cells were passaged approximately twice a week. The MDA-MB-231 and MDA-MB-468 cells used in experiments were derived from passage numbers 5e10 (P5eP10) to prevent significant variation between experiments.

C. nutans bioactive compound identification and extract preparation
The C. nutans ethanolic leaf extract was prepared in the Integrative Medicine Laboratory at the Advanced Medical and Dental Institute, Universiti Sains Malaysia (USM), Malaysia. The extract was subjected to various quality controls, as reported earlier. 29,30 The plant was authenticated by its voucher specimen number 11465, and placed at the Herbarium Unit, School of Biological Sciences, USM, Malaysia. 29,30 The leaves were first sorted, dried, and pulverized. The powder (255 g) was then macerated in 1L of ethanol in a conical flask at room temperature for three days with frequent stirring with a magnetic stirrer. The mixture was then filtered, and fresh ethanol was added to the residue. This procedure was performed several times until a clear and colorless solution was achieved. The filtrates were then mixed and evaporated under reduced pressure at 40 C using a rotary evaporator (BUCHI Rotavapor R-200, Switzerland). Following ethanol evaporation, the dried extract was lyophilized at À50 C in a freeze drier (CHRIST Alpha 1e4 LD plus Freeze Dryer, Germany) to remove all traces of moisture. The phytochemical fingerprint of C. nutans leaf extract was evaluated using LCeMS/MS analysis and a dual ESI source for both positive and negative ion mode analysis. The experiments were conducted to identify the presence and percentage of bioactive compounds in the extract. This was determined by the intensity arriving from the mass spectra of LCeMS/MS. To prepare the extract for treatment, a stock solution of aqueous extract was prepared by dissolving 100 mg of C. nutans extract in 1 mL of 10% Tween 20. Thus, the initial concentration of the aqueous stock solution was 100 mg/mL. The initial concentration of C. nutans was further diluted with serum-free media into several different concentrations (2.5, 5, 10, 20, 30, and 50 mg/ mL) and further used to study anticancer effects on MDA-MB-231 and MDA-MB-468 breast cancer cells.

The interaction between cisplatin and C. nutans
The drugedrug interaction of the combined cisplatin and C. nutans treatment (fixed cisplatin concentration (3.05 mg/ mL) followed by different concentrations of C. nutans (2.5, 5, 10, 20, 30 and 50 mg/mL)) was accomplished by conducting the isobologram-combination index analysis using Compu-Syn software. The average inhibition effect, fa, of each treatment was calculated based on the median effect equation: fa/fu ¼ (D/Dm) m. The inhibitory effect was determined based on cell viability data and the combination index (CI) for each treatment. The drugedrug interaction effect was demonstrated either as an antagonistic effect (CI value more than 1.0; additive effect: CI value ¼ 1.0) or a synergistic effect (CI value less than 1.0).

Detection of caspase-3/7 activity
Both MDA-MB-231 and MDA-MB-468 cells were treated with 20 nM Taxol (positive control) as well as IC 50 concentrations of cisplatin, C. nutans, and a combination of cisplatin and C. nutans. Apoptotic activity was assessed via the Caspase-GloÒ 3/7 Assay (Promega, USA) according to the manufacturer's instructions Luminescence measurements were read using a GloMaxÒ-Multi Detection System (Promega, USA). Caspase-3/7 activity was expressed as the fold change in enzyme activity over untreated cells (control). All treatments were compared to untreated (control) cells, which were normalized to 1-fold of caspase-3/7 activity. The experiment was completed in triplicate to obtain average results.

Detection of cell invasion
The MDA-MB-231 cells were treated with IC 50 concentrations of cisplatin, C. nutans, a combination of cisplatin and C. nutans. The metastatic ability of MDA-MB-231 breast cancer cells upon cisplatin, C. nutans and combined cisplatin and C. nutans treatments using IC 50 concentrations, were detected using the CultrexÒ BME Cell Invasion Assay (Trevigen) in accordance with the manufacturer's instructions. The fluorescence intensity was read at 485 nm excitation and 520 nm emission using a GloMaxÒ-Multi Microplate Multimode Reader (Promega, USA). The combined cisplatin and C. nutans treatment consisted of cells treated with cisplatin (3.05 mg/mL for 24 h) followed by C. nutans (10 mg/mL for 24 h). The invading cells were quantified based on metastatic index: (Metastatic index ¼ (fluorescence reading (treatment))/(fluorescence reading (negative control)) Â 100%). The experiment was completed in triplicates to obtain average results.

Quantitative real time polymerase chain reaction (qRT-PCR)
MDA-MB-231 cells were lysed using the QIAzolÒ Lysis Reagent (Qiagen, Germany). Total cellular RNA was then extracted using an RNA Clean & ConcentratorÔ-5 Kit (Zymo Research, USA). RNA quantity and quality were assessed with a NanoDrop 1000 (NanoDrop, Wilmington, DE, USA). Next, cDNA was synthesized from RNA using an iScript Synthesis Kit (Bio-rad, USA).
PCR reactions were then incubated in a Bio-rad iCycler TM Optical Module (Bio-rad, USA) at 42 C for 30 min and 85 C for 5 min to synthesize complementary DNA (cDNA) by reverse transcription. The final concentration of the cDNA product per sample was 20 ng/mL. Next, a mixture consisting of specific cDNA (1 mL), 2Â SsoAdvancedÔ universal SYBRÒ Green supermix (10 mL) (Bio-rad, USA) and nuclease-free water (9 mL) were added into a customized skirted 96-well PCR plate with specific targeted genes ( Table 2) to detect the relative gene expression before and after drug treatment. Universal SYBRÒ Green supermix was used in the qRT-PCR assay to assist in the fluorescent signaling of gene expression levels.
In addition, 1 mL of PCR control assay template was added into the positive control well. The PCR reactions were then incubated in a Bio-rad iCycler TM Optical Module (Bio-rad, USA) using a reaction protocol of 95 C for 2 min (1 cycle for activation step), 95 C for 5 s (40 cycles for denaturation step), 60 C for 30 s (40 cycles for annealing/ extension step), and 65e95 C for 5 s (1 step for melt curve). Positive controls included PCR control assay templates (RQ1 and RQ2 (for RNA integrity)) and RT (for reverse transcriptase), while gDNA (genomic DNA contamination) was used as a negative control. Each PCR reaction was normalized to GAPDH (housekeeping gene) and plotted as relative mRNA expression. Experiments were performed in triplicate in two independent experiments. Bars represent mean AE SEM (n ¼ 2). *p 0.05, **p 0.005, ***p 0.0005, ****p 0.0001.

Statistical analysis
Analyses of the results were performed with Microsoft Excel and GraphPad Prism version 9 (GraphPad Software, Inc.). The Student's paired t-test with a two-tailed distribution was used to compare cisplatin, C. nutans and a combination of cisplatin and C. nutans and untreated MDA-MB-231 and MDA-MB-468 breast cancer cells. The results are presented as mean AE SEM. The significance is shown as: *p 0.05; **p 0.005; ***p 0.0005; ****p < 0.0001.

Results
Bioactive compounds of C. nutans with anticancer properties The phytochemical fingerprint of C. nutans leaf extract was evaluated by LCeMS analysis using a dual ESI source for both positive and negative ion mode analysis. These experiments were conducted to determine the percentage of bioactive compounds present in the extract by determining the intensity derived from the mass spectra of LCeMS/MS ( Table 3). The putatively identified compounds with positive ionization were homoarecoline (9.96%), C16 sphinganine A total of 18 major bioactive compounds with potential anticancer properties were identified by ethanol extraction. 31e39 Of these, fatty acid derivatives (28.34%),   (Figure 1BeC). In contrast, the proliferative capacity of MDA-MB-468 cells exhibited a greater inhibitory effect upon cisplatin and C. nutans treatment, respectively, ranging from 13 to 14.37% at 2.5 mg/mL of C. nutans and 0.76 mg/mL of cisplatin, 32e44.2% at 10 mg/mL of C. nutans and 3.05 mg/ mL of cisplatin, and over 70% at 50 mg/mL of C. nutans and 15.23 mg/mL of cisplatin (Figure 2BeC).
In contrast to the singular treatments with cisplatin and C. nutans, the combined cisplatin and C. nutans treatment had a more potent effect on cell viability and proliferation inhibition in both TNBC subtypes. A fixed 3.05 mg/mL cisplatin treatment for 24 h prior to various C. nutans treatments,    proliferation ranged between 13 and 15% for cisplatin-2.5 mg/ mL C. nutans, 24e26% for cisplatin-5 mg/mL C. nutans, 45e 46% for cisplatin-10 mg/mL C. nutans, 51e62% for cisplatin-20 mg/mL C. nutans, 66e69% for cisplatin-30 mg/mL C. nutans and 76e78% for cisplatin-50 mg/mL C. nutans when compared to singular 2.5 mg/mL C. nutans (0e4%), 5 mg/mL C. nutans (15e17%), 10 mg/mL C. nutans (24e29%), 20 mg/mL C. nutans (30e38%), 30 mg/mL C. nutans (42e50%) and 50 mg/ mL C. nutans (49e59%), as shown in Figure 1FeG and summarized in Table 5. 13%, 12e20%, 20e32%, 28e41%, 37e63% and 48e73%, respectively, as compared to singular C. nutans treatment at the same concentrations (15e16%, 26e30%, 57e59%, 61e 65%, 69e72% and 72e77%) respectively (Figure 2EeG; Table 5). In addition, the combined treatment also significantly reduced the IC 50    Based on the findings in Figures 1 and 2, it appeared that the singular cisplatin and C. nutans treatments potentially exerted a different mechanism of action when compared to the combined treatment. Hence, we investigated the ability of each treatment to induce apoptosis via Caspase 3/7 activity in TNBC cells represented by MDA-MB-231 and MDA-MB-468 cells (Figure 4). Figure 4 shows that the activation of Caspase-3/7 was negligible in cells treated with IC 50 of cisplatin and C. nutans concentrations in contrast to treatment with 20 nM Taxol (1.28-fold and 1.18fold) and combined treatment (3.05 mg/mL cisplatin þ 10 mg/ mL C. nutans) by 2.73-fold and 3.53-fold, respectively. This finding can be supported by the morphological changes in MDA-MB-231 and MDA-MB-468 cells upon combined treatment, which appeared unhealthy, rounded, uneven, and apoptotic-like ( Figures 1D and 2D) in comparison to the healthy and viable untreated cells (control) (Figures 1A  and 2A). This cells also appeared morphologically different to those receiving single cisplatin and C. nutans treatments (Figure 1BeC and Figure 2BeC).

Inhibition of cell invasion by MDA-MD-231 breast cancer cells when treated with a combination of cisplatin and C. nutans
MDA-MB-231 cells are a highly metastatic breast cancer subtype. 40 Hence, we studied the ability of cisplatin, C. nutans and the combination of cisplatin and C. nutans to inhibit the metastatic capacity of MDA-MB-231 cells. We found that MDA-MB-231 cells exhibited reduced metastatic capacity but at different capacities; the highest level of inhibition was observed in cells treated with the combination of cisplatin and C. nutans when compared to moderate inhibition upon singular cisplatin and C. nutans treatment ( Figure 5). Figure 5 shows that the lowest level To study the role of cisplatin, C. nutans and the combination of cisplatin and C. nutans on gene regulation, we next investigated the expression of several genes associated with breast cancer stem cells (integrin alpha 6, ITGA6, CD49f), metastatic markers (Kruppel-like Factor 4, KLF4), and differentiation markers (cytokeratin-18, KRT18; Tubulin alpha 1A, TUBA1A) ( Figure 6). All treatments were carried out using the IC 50 concentration of each drug prior to gene expression studies. Figure 6 clearly shows that MDA-MB-231 cells expressed all of the genes studied; the expression levels of these genes were affected by treatment with cisplatin, C. nutans and the combination of cisplatin and C. nutans treatments, respectively. An obvious and significant upregulation of KLF4 mRNA expression was exhibited in MDA-MB-231 with all treatments (cisplatin, 6.256; C. nutans, 10.214; combination, 8.436) when compared to the expression levels of other genes. Similarly, the mRNA expression of both KRT18 and TUBA1A were also up-regulated upon single cisplatin and C. nutans treatments; however down-regulation was observed in KRT18 expression (39%) along with an upregulation of TUBA1A expression (19%) with the combined cisplatin and C. nutans treatment. In contrast to the increased mRNA expression levels of KLF4, the expression levels of CD49f were down-regulated in response to cisplatin (19%) and combined cisplatin and C. nutans (22%) treatments. However, a significant increase in CD49f expression was observed in response to single C. nutans treatment. A down-regulation of CD49f, a specific marker of breast cancer stem cells, was correlated with a reduction in cancer cell stemness, reduced proliferative capacity, and the induction of differentiation.

Discussion
Current research focuses on targeting and eliminating the resistant or CSC sub-population within a tumor, especially for difficult-to-treat cancers, including Triple Negative Breast Cancer (TNBC). This invasive form of cancer is often correlated with a poor prognosis and low survival rates due to its highly metastatic nature and the lack of targeted therapies. 41,42 These cancers are sensitive to cisplatin treatment; however, at high doses, cisplatin often leads to severe side effects, 17,43 in addition to the development of tumor cell resistance to cisplatin. Therefore, the use of combined therapy with cisplatin is recommended to potentially target and kill the CSCs that are responsible for the progression and metastasis of TNBCs. 44 It is evident that various phytochemicals or plant-derived compounds can be used in combination with cisplatin and enhance the efficiency of this drug; this practice can also reduce drug concentrations and the toxicity induced by cisplatin. 11,45 In this investigation, we demonstrated that both MDA-MB-231 (a highly metastatic and poorly differentiated cell line) and MDA-MB-468 (a metastatic and less differentiated cell line) that represent a TNBC subtype, were sensitized towards cisplatin by supplementation with an ethanolic leaf extract of C. nutans known to be enriched with compounds possessing anticancer properties. 31e39, 46 This sensitization can be proven via the achievement of a lower cisplatin IC 50 concentration by 50% upon the treatment of addition of MDA-MB-231 cells with C. nutans ( Figure 1H). In addition, the metastatic potential of MDA-MB-231 cells was significantly inhibited by almost 50% when treated with 3.05 mg/mL cisplatin þ 10 mg/mL C. nutans when compared to 3.05 mg/mL cisplatin treatment. The presence of various bioactive compounds in C. nutans extract appears to overcome cisplatin resistance by interfering with  specific signaling pathways that are crucial for CSC maintenance. 47 These crucial signaling pathways are known to either positively regulate differentiation and apoptosis or deactivate pathways that are responsible for proliferation and metastasis. In this study, a number of bioactive compounds, including oleamide, carboxylic acid esters, and glucosides, were identified to have potential anticancer activities. Fatty acid derivatives, such as oleamide, have been shown to exert significant anticancer properties by hindering cell proliferation in various cell lines, 34,48e50 including MDA-MB-231 cells. 51 Carboxylic acid esters that are present in plants, including C. nutans, play an important role as bioactive compounds in various cancer cell lines. The presence of carboxylic acid ester groups in synthesized cisplatineacridine hybrids has been associated with the significant inhibition of cell proliferation in ovarian and breast cancer cells. 52e54 The third most abundantly present bioactive compound of C. nutans, glycosides, are commonly found in many plants and have demonstrated strong cytotoxic effects in various cancer cell lines. 55,56 The anticancer of C. nutans was facilitated by numerous mechanisms. For example, flavonoid glycosides exerted obvious anticancer activity in HeLa cells via an apoptosis mechanism. 57 In addition, paeoniflorin, a representative of glycoside, showed antitumor effects on a diverse range of tumors, both in vivo and in vitro, including breast cancer via the induction of tumor cell apoptosis in addition to the inhibition of proliferation, tumor invasion and metastasis. 58 It is most likely that the abundance of oleamide, carboxylic acid esters, and glucosides, in C. nutans extracts is responsible for the enhanced inhibitory effects in MDA-MB-231 and MDA-MB-468 cells. However, it is important to further investigate each bioactive compound present in C. nutans extracts and correlate these with anticancer properties via the treatment of MDA-MB-231 and MDA-MB-468 cells with a combination of cisplatin and C. nutans.
When correlating the ability to inhibit cell viability and proliferation of MDA-MB-231 and MDA-MB-468 cells, it can be postulated that both cisplatin and C. nutans treatments were able to limit the proliferative capacity, although cells were viable post-treatment (Figure 1DeE and Figure 2DeE). This may suggest that singular cisplatin and C. nutans may have induced the differentiation of MDA-MB-231 and MDA-MB-468 cells based on morphological changes (enlarged and elongated cells; thinner/smaller and elongated cells), as seen in Figure 1BeC and Figure 2Be C, respectively. Cisplatin is commonly known to interfere with DNA activity in tumors by the formation of adducts (inter-strand or intra-strand crosslinks) and consequently preventing the DNA repair process, thus leading to apoptosis. 59,60 However, it appears that cisplatin may have a different mode of action in response to resistant CSCs. A previous study showed that cisplatin is able to induce differentiation in TNBCs enriched with CSCs 1 but lacks therapeutic efficacy as a single anticancer agent. 11,44 This differentiation by cisplatin is presumed to be caused by the binding of cisplatin compounds to other target molecules apart from nuclear DNA, including some peptides, cellular proteins, and RNAs. 16,61,62 The binding of cisplatin to these targets could also lead to epigenetic modifications by switching on and/or off the expression of specific epigenetic biomarkers related to stemness and differentiation. 63,64 In contrast, as yet, no researchers have correlated C. nutans and the differentiation of cancer cells. Nevertheless, studies have shown that oleamide, a fatty acid derivative, plays important roles as a gap junction modulator and antiangiogenic agent, thus resulting in reduced metastases and invasion. 51,65 Some gap junction proteins assist in intracellular communication between tumor cells and causes these cells to go undetected by the immune system, thus leading to cancer metastases and progression. 66e68 Previous studies have shown that the gap junction proteins Connexin-26 (Cx26) and Connexin-43 (Cx43) are often upregulated and downregulated, respectively, in the CSC population in TNBCs which are responsible for driving selfrenewal. 47,66,68,69 Oleamide is known to modulate the function of gap junction proteins via negative and positive regulation by interrupting the intracellular communication between tumor cells. These regulatory events consequently lead to the structural inhibition of gap junction channels 51,66 and the sensitization of CSCs to certain chemotherapeutic drugs, such as cisplatin by improving its uptake. 68,70 However, some studies have contradicted these findings by observing that oleamide increased survival and proliferation upon combined treatment in manner that depended on the type of cancer cell and the anticancer agent used together with oleamide. 71,72 Furthermore, butanedioic acid, a carboxylic acid ester, is known to be a metabolic suppressor and can lead to reduced proliferation and stemness. 73,74 In short, the ethanolic extract of C. nutans, which is enriched with fatty acids, carboxylic acid esters (long chain fatty acids), glycosides, and their derivatives, are presumed to be the major contributing bioactive compounds exerting anti-cancerous activities through multiple pathways. 34,75e78 In comparison to single treatments, the combined treatment of cisplatin and C. nutans on MDA-MB-231 and MDA-MB-468 cells significantly promoted a synergistic anticancer effect with increasing C. nutans concentration. The combined treatment at a lower C. nutans concentration (fixed at 3.05 ug/mL of cisplatin and C. nutans at 2.5 and 5 ug/mL) exerted less cytotoxic effects in both of the cell lines tested. It is presumed that at low doses, C. nutans is not capable of inducing a significant inhibitory effect but reacts in an antagonistic manner. 79,80 However, a clear shift from antagonistic to synergistic effects was observed with increasing concentrations of C. nutans. The presence of a high fatty acid content in the extract of C. nutans leaves that are lipophilic in nature could be a contributing factor in increasing the bioavailability and uptake of cisplatin into tumor cells in addition to its own anticancer properties. 34,81 Although cisplatin is known for its potent anticancer activity on various solid tumors, the cisplatinplasma membrane interaction is often correlated with poor uptake and resistance. 82,83 Consequently, a higher dosage may be required; however, this would, in turn, cause adverse side effects in patients. The presence of fatty acids in the form of oleamide, as the major bioactive compound in C. nutans leaf extract, in combination with cisplatin, appears to complement the uptake of cisplatin more efficiently into tumor cells and in addition interacts with cisplatin to produce synergistic anticancer effects.
In addition, the combined treatment also significantly reduced the cell viability and proliferation by more than 70%; the remaining cells were partially dead or undergoing early apoptosis ( Figures 1D, G, 2D and G). Figure 4 confirms this claim as a significant induction of caspase 3/7 activity was exhibited in the remaining cisplatineC. nutans treated cells when compared to Taxol (the positive control) as well as cells treated with singular cisplatin and C. nutans. The seemingly reduced cell viability and proliferation at a similar percentage in the combined treatment also suggest that the reduction is due to cell death, which correlates well with the activation of caspase-3/7 activity (Figures 1D, G,  2D, G and 4). This interaction between the major bioactive compounds in C. nutans and cisplatin sensitized resistant TNBC cells to cisplatin and ultimately initiated apoptosis in TNBC cells. The negligible apoptotic induction in single cisplatin and C. nutans treated cells in contrast, may suggest that the remaining cells did not undergo apoptosis but possibly were differentiated instead; this could have led to the compromised stemness of cancer cells, along with limited proliferative and metastatic capacity 84 in MDA-MB-231 cells, as demonstrated by the reduced metastatic index and the downregulation and upregulation of breast CSCs and differentiation markers, respectively (Figures 5e6).
Cisplatin treated MDA-MB-231 cells exhibited a downregulation in the mRNA expression of CD49f (a breast CSC marker) and an upregulation of TUBA1A and KRT18 (neural and luminal epithelial differentiation markers). With regards to C. nutans treated MDA-MB-231 cells, CD49f mRNA expression was upregulated along with KRT18 and TUBA1A. This finding may suggest that C. nutans did not interfere with the expression of CD49f at the gene level. However, it would be interesting to investigate the expression of CD49f protein as C. nutans could induce epigenetic modification of CD49f expression at the post-translational level. The findings from our gene expression study support the previous claims made based on Figures 1e3 in which the single treatments potentially induced differentiation. In addition to CD49f, KRT18 and TUBA1A, the expression of KLF4 was significantly higher than the expression levels of other selected genes (CD49f, KRT18, and TUBA1A) upon single cisplatin and C. nutans as well as combined cisplatin and C. nutans treatments. KLF4 is an embryonic stem cell (ESC) marker and a transcription factor (TF) that is expressed at high levels by most breast cancer cell types. 85,86 KLF4 acts as a transcriptional factor responsible for epithelial cell proliferation and differentiation. 87 Generally, KLF4 has been considered as a negative cell cycle regulator, monitoring multiple genes to promote and inhibit proliferation. 87 KLF4 has been recognized as one of the "pluripotency genes" due to its ability of induce pluripotent stem cells. 87,88 KLF4 is able to reprogram normal somatic cells into stem-like cells to maintain its ability to self-renew and prevent differentiation. 87,89 Despite KLF4 being an early-stage ESC and a TF that aids in the maintenance of the embryonic stage in cancer cells, the expression of KLF4 in TNBCs may act as a gene that suppresses tumor growth by targeting CSCs. This finding can be supported by a few recent findings which claim that KLF4 can have dual functions in which it either acts as a potent oncogenic activator or a tumor suppressor, as determined by the type of cancer and its microenvironment. 3,88,90 Previous studies showed that high levels of KLF4 expression in TNBC patients correlated with better overall survival and cancer-free survival rate 42 and also correlated with the sensitization of cancer cells to cisplatin and paclitaxel treatment. 91 An earlier study by Zhang et al., in 2010 showed that KLF4 interacts with NANOG, another TF, to prevent ESC differentiation; however, the knockdown of NANOG resulted in differentiation. 92 This argument can be well correlated with MDA-MB-231 cells, as these invasive TNBC cells are enriched with CSCs but lack NANOG gene expression. 93 Therefore, the lack of NANOG expression by MDA-MB-231 cells may have switched the oncogenic KLF4 to tumor suppressor mode, which consequently induced differentiation upon cisplatin and C. nutans treatment. With regards to KLF4 gene up-regulation and the induction of differentiation, previous studies have claimed that KLF4 specifically induces the differentiation of epithelial cell origin 92,94 which again may explain the correlation of KLF4 and the differentiation induction of MDA-MB-231 cells which are epithelial in origin. 95 In comparison to the single cisplatin and C. nutans treatments, the combined treatment downregulated CD49f and KRT18 expression; this may have arisen as the number of apoptotic cells increased.
Collectively, these findings suggest that the synergistic effect of combined cisplatineC. nutans treatment is closely related to the interactions of oleamide, the major bioactive compound in C. nutans. Oleamide modulates gap junction proteins and may help to sensitize MDA-MB-231 and MDA-MB-468 cells by upregulating Cx43 which has been shown to increase the uptake of cisplatin 70 and promote apoptosis while downregulating Cx26 which is responsible for metastasis and cancer progression. 68,96 This is interesting because gap junction communication plays an important role in calcium (Ca 2þ ) signaling whereas cisplatin uptake, along with its cytotoxicity and chemosensitivity, are dependent on the cytosolic calcium concentration. 97 This claim can be supported by the lower cisplatin and IC 50 concentrations required to exert significant anticancer activity in MDA-MB-231 and MDA-MB-468 cells apart from apoptotic induction and the limited metastatic ability via the upregulation of differentiation and the downregulation of CSC makers. However, the mechanisms by which oleamide can induce connexins to mediate cisplatin chemosensitivity remain unclear and need to be investigated further.
In summary, the combination of cisplatin and phytochemicals, such as C. nutans, could be a potential therapeutic option for treating and managing TNBCs and possibly other types of cancers that are enriched with CSCs. Figure 7 shows a proposed model based on our findings, which highlights the possible mechanism of action exerted by the combined treatment of TNBC cells with cisplatin and C. nutans.

Conclusion
To conclude, both cisplatin and C. nutans were found to be potent anticancer agents and induced the differentiation of tumor cells as a single anticancer agent while promoting cell death via apoptosis upon combined cisplatin and C. nutans treatment which targeted the more differentiated MDA-MB-231 and MDA-MB-468 cells. Gene expression studies also revealed that cisplatin and C. nutans differentially regulated specific genes in MDA-MB-231 TNBC cells. Although the exact mechanism of the cisplatin and C. nutans combination remains unclear, we showed that each of the anticancer agents may possess multiple mechanisms of action. We also demonstrated that C. nutans leave extract is enriched with fatty acids, which may have acted as a drug carrier for cisplatin uptake into tumor cells more efficiently in addition to the synergistic interactions of cisplatin and C. nutans, thus leading to apoptotic induction. Further investigations are now required to gain a better understanding of how the cisplatin and C. nutans combination interacts with s as well as other solid tumor microenvironments.

Conflict of interest
The authors declare that there are no conflicts of interest.

Ethical approval
No ethical approval was required as this study did not involve human participants or laboratory animals.